Huntington's Disease is an inherited, autosomal dominant neurological disease. It is uncommon, affecting approximately 1 in 10000 individuals (Breighton and Hayden 1981). The disease does not usually become clinically apparent until the fifth decade of life, and results in psychiatric disturbance, involuntary movement disorder, and cognitive decline associated with inexorable progression to death, typically 17 years following onset.
The gene responsible for Huntington's disease is called huntingtin. It is located on chromosome 4p, presenting an effective means of preclinical and antenatal diagnosis. The genetic abnormality consists in an excess number of tandemly repeated CAG nucleotide sequences.
The huntingtin gene is ubiquitously expressed (Strong et al. 1993) and conserved across a wide range of species (Lin et al., 1994). Structural analysis of its promoter region is consistent with it being a housekeeping gene (Lin et al., 1995). The huntingtin gene encompasses 67 exons, spans over 200 kb (Ambrose et al., 1994) and is associated with two transcripts of 10.3 kb and 13.6 kb, differing with respect to their 3′ untranslated regions (Lin et al., 1993). Both messages are predicted to encode a 348 kilodalton protein containing 3144 amino acids. In addition, the huntingtin gene encompasses a highly polymorphic CAG repeat, which varies in number from 8 to 35 in normal individuals (Kremer et al., 1994). CAG expansion beyond 36 CAG repeats is seen in persons with Huntington's disease.
The increase in size of the CAG repeat in persons with Huntington's disease shows a highly significant correlation with age of onset of clinical features. This association is particularly striking for persons with juvenile onset Huntington's disease who have very significant expansion, usually beyond 50 repeats. The CAG repeat length in Huntington's disease families does exhibit some instability that is particularly marked when children inherit the huntingtin gene from affected fathers.
In HD, it is not known how this widely expressed gene results in selective neuronal death. Furthermore, sequence analysis revealed no obvious homology to other known genes and no structural motifs or functional domains were identified which clearly provide insights into its function. In particular, the question of how these widely expressed genes cause selective neuronal death remains unanswered.
The major site of pathology in HD is the striatum, where up to 90% of the neurons may be depleted. Within the striatum there is a selective loss of certain neuronal populations. Striatal medium-sized spiny neurons, which contain the neurochemical markers gamma-aminobutyric acid (GABA), substance P, dynorphin, and enkephalin are preferentially affected. In contrast, medium-sized aspiny neurons containing the neuropeptides somatostatin and neuropeptide Y, and large aspiny neurons containing choline acetyltransferase (ChAT) activity, are spared (despite an overall loss of ChAT activity). Dopaminergic and serotonergic afferent projections are also spared. (Beal et al, 1991).
The impaired cognitive functions and eventual dementia may be due either to the loss of cortical neurons or to the disruption of normal activity in the cognitive portions of the basal ganglia, namely the dorsolateral prefrontal and lateral orbitofrontal circuits. The characteristic chorea is believed to be caused by the neuronal loss in the striatum, although a reduction in subthalamic nucleus activity may also contribute to it. Normally a balance is maintained among the activities of three biochemically distinct but functionally interrelated systems: (1) the nigrostriatal dopaminergic system; (2) the intrastriatal cholinergic neurons; and (3) the GABA-ergic system, which projects from the striatum to the globus pallidus and substantia nigra. An imbalance anywhere in the dopamine, acetylcholine, or GABA systems can cause involuntary movements. Both choline acetyltransferase, the enzyme required for the formulation of acetylcholine, and glutamic acid decarboxylase, the enzyme required to synthesize GABA, are markedly decreased in the striatum of patients with HD. These enzyme deficits are consistent with the clinical observation that choreic movements worsen in patients with HD following administration of L-DOPA.
Glutamate-induced neuronal cell death is believed to contribute to Huntington's disease. Glutamate is the principal excitatory transmitter in the brain. It excites virtually all central neurons and is present in the nerve terminals in extremely high concentrations (10−3 M). Glutamate receptors are divided into four types (named after their model agonists): kainate receptors, N-methyl-D-aspartate (NMDA) receptors, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors, and metabolotrophic receptors. Many normal synaptic transmission events involve glutamate release.
Glutamate can also induce neurotoxicity and neuronal death at high levels (Choi, 1988). The mechanism of cell death occurs primarily by the persistent action of glutamate on the N-methyl-D-aspartate (NMDA) type of glutamate receptors and the resulting excessive influx of Ca2+. The excessive Ca2+ mobilizes active Ca2+-dependent proteases and activates phospholipase A2, which in turn liberates arachidonic acid, leading to the production of substances causing inflammation and free radicals that can trigger further destructive events. These toxic changes produced by glutamate, called glutamate excitotoxicity, are believed to be the cause of cell damage and death after acute brain injury such as stroke or excessive convulsions. It has been suggested that excitotoxicity may be involved in brain ischemia, Alzheimer's disease and HD (Greenamyre et al, 1985; Choi et al, 1988).
Several animal models mimicking HD pathology have been set up. Injection of glutamate receptor agonists into rat striatum can produce a pattern of neuronal cell loss similar to HD. Although the majority of the neurons within the actual injection site die, there is a surrounding gradual transition zone that exhibits selective cell death. Initial studies with kainic acid (KA)-induced lesions showed a striking resemblance to HD. KA is isolated from the seaweed Diginea simplex and is not found in the mammalian brain. Intrastriatal injections of KA result in neuronal loss and gliosis, with reductions in markers of intrinsic striatal neurons, yet a preservation of dopaminergic afferents. These KA-induced lesions, however, are an imperfect model of HD because they result in a significant depletion of somatostatin levels and a loss of somatostatin neurons. Lesions produced by NMDA receptor agonists such as quinolinic acid (QA) provide a better model of HD, because they result in relative sparing of somatostatin and neuropeptide Y levels, despite significant depletions of both GABA and substance P levels. Long-term (6 months to 1 year) follow-up of QA lesions reveals increases in somatostatin and neuropeptide Y and in serotonin and in 5-hydroxyindoleacetic acid (HIAA), which are similar to the findings in actual HD patients. Chronic QA lesions therefore closely resemble the neurochemical features of HD (Beal et al, 1991.) Others have confirmed that QA-induced injury of the striatum can resemble the histopathology of HD (See, e.g., Roberts et al, 1993).
These animal models have been extensively used to develop strategies that may be relevant for the treatment of HD, such as cell replacement and neuroprotective approaches. A significant rescue of degenerating GABAergic neurons was observed following the grafting of fetal striatal cells or the administration of neurotrophic factors in QA-lesioned rats (Bemelmans et al. 1999).
Further neurochemical abnormalities have been identified in HD, for example reduced levels of choline acetyltransferase and gamma aminobutyric acid in the basal ganglia. These changes are presumable secondary to the primary neuronal loss.
There is presently no cure for Huntington's disease. The choreic movements and agitated behaviors may be suppressed, usually only partially, by antipsychotics (e.g., chlorpromazine 100 to 900 mg/day per os or haloperidol 10 to 90 mg/day per os) or reserpine begun with 0.1 mg/day per os and increased until adverse effects of lethargy, hypotension, or parkinsonism occur. Therapeutic strategies to replace brain GABA stores have been ineffective. Experimental therapies aim to reduce glutamatergic neurotransmission via the N-methyl-D-aspartate receptor and bolster mitochondrial energy production. Long-term clinical trials are needed to assess these therapies
All treatment presently available focuses on addressing the disease's symptoms, preventing associated complications and providing support and assistance to the patient. For those diagnosed with HD, physicians often prescribe various medications to help control emotional and movement problems. Benzodiazepines may alleviate choreic movements, and antipsychotic drugs may help control hallucinations, delusions or violent outbursts. If the patient suffers from depression, the physician may prescribe antidepressants. Tranquilizers can be used to treat anxiety, and lithium may be prescribed for patients who exhibit pathological excitement or severe mood swings. Other medications may be prescribed for the severe obsessive-compulsive behaviors some individuals with HD develop.
Therefore, there is an unmet need for a medicament, pharmaceutical compositions and methods useful for the treatment of Huntington's disease. Such medicaments, pharmaceutical compositions and methods will ideally stop the progression of the degenerative disease and even promote regeneration of the damaged neurons, without severe adverse side effects.
Several neurotrophic factors have been tested in animal models of HD so far (Andersen et al, 1996). Brain-derived growth factor (BDNF), nerve growth factor (NGF) or neurotrophin-3 (NT-3) did not result in protection of striatal neurons from QA induced cell death. Ciliary neurotrophic factor (CNTF) had some protective effect in a monkey model of HD (Emerich et al, 1997).
Some neuroprotective strategies using gene therapeutic approaches have been suggested. These approaches rely on the development of effective delivery systems leading to robust expression of the transgene over extended periods of time and the presence of therapeutic protein in large area of the striatum. The transplantation of genetically engineered cells, the implantation of encapsulated cells releasing neurotrophic factors and more recently an in vivo gene therapy approach with an adenoviral vector have been tested (Emerich et al. 1996, Bemelmans et al. 1999). HIV-1-derived lentiviral vectors have recently emerged as a promising gene delivery system in the CNS (Naldini et al. 1996a; Klimatcheva et al. 1999). Since 1996, significant efforts have been dedicated to increase the safety of the system and to define the minimal genetic information required for the transduction HIV-1 vectors.
To minimize the risk of emergence of replication-competent recombinants so-called SIN (self inactivating) vectors were developed. The SIN design results in the deletion of the U3 region in the long terminal repeat (LTR) from the transfer vector, removing the major part of the viral transcriptional elements prior to integration. This modification not only reduces the risk of appearance of replication-competent viruses through recombination, but also eliminates transcriptional interference between the LTR and the internal promoter, and minimizes the chance that genes adjacent to the vector integration site become activated (Déglon et al., 2000).
This expression vector system has been previously demonstrated to lead to a high and consistent transduction of neuronal cells with a SIN expressing the LacZ reporter gene in mice, rats and primates (Bensadoun et al. 2000; Déglon et al. 2000; Kordower et al. 1999). In addition, the presence of the post-transcriptional element from the woodchuck hepatitis virus (Zufferey et al. 1999) was shown to result in a 3-4 fold increase of the transgene expression level (Déglon et al, 2000) similarly to what was observed in adeno-associated viruses (Loeb et al. 1999).
Experiments on the effects of a cytokine, interleukin-6 (IL-6), on cells of the central and peripheral nervous system indicate that IL-6 may have protective effects on neuronal cells as well as some impact on inflammatory neurodegenerative processes (Gadient and Often, 1997, Mendel et al, 1998). IL-6 was found to prevent glutamate-induced cell death in hippocampal (Yamada et al., 1994) as well as in striatal (Toulmond et al., 1992) neurons. The IL-6 mechanism of neuroprotection against toxicity elicited by NMDA, the selective agonist for NMDA subtype of glutamate receptors, is still unknown. In fact IL-6 was found to enhance the NMDA-mediated intracellular calcium elevation. In transgenic mice expressing high levels of both human IL-6 and human soluble IL-6R (sIL6-R), an accelerated nerve regeneration was observed following injury of the hypoglossal nerve as shown by retrograde labeling of the hypoglossal nuclei in the brain (Hirota et al, 1996). Recently, there has been some evidence that IL-6 is implied in a neurological disease, the demyelinating disorder Multiple Sclerosis (MS) (Mendel et al., 1998). Mice lacking the IL-6 gene were resistant to the experimental induction of the disease.
Interleukin-6 (IL-6) is a well known cytokine whose biological activities are mediated by a membrane receptor system comprising two different proteins one named IL-6 Receptor (IL-6R or gp80) and the other gp130 (reviewed by Hirano et al, 1994). Soluble forms of IL-6R (sIL-6R), corresponding to the extracellular domain of gp80, are natural products of the human body found as glycoproteins in blood and in urine (Novick et al, 1990, 1992). An exceptional property of sIL-6R molecules is that they act as potent agonists of IL-6 on many cell types including human cells (Taga et al, 1989; Novick et al, 1992). Even without the intracytoplasmic domain of gp80, sIL-6R is still capable of triggering the dimerization of gp130 in response to IL-6, which in turn mediates the subsequent IL-6-specific signal transduction and biological effects (Murakanni et al, 1993). sIL-6R has two types of interaction with gp130 both of which are essential for the IL-6 specific biological activities (Halimi et al., 1995), and the active IL-6 receptor complex was proposed to be a hexameric structure formed by two gp130 chains, two IL-6R and two IL-6 ligands (Ward et al., 1994; Paonessa et al, 1995).
Chimeric molecules linking the soluble IL-6 receptor and IL-6 together have been described (Chebath et al., 1997. Fischer et al., 1997. WO 99/02552 and WO 97/32891). They have been designated IL-6R/IL-6 chimera and Hyper-IL-6, respectively. The chimeric IL-6R/IL-6 molecules were generated by fusing the entire coding regions of the cDNAs encoding the soluble IL-6 receptor (sIL-6R) and IL-6. Recombinant IL-6R/IL-6 chimera was produced in CHO cells (Chebath et al, 1997, WO99/02552). The IL-6R/IL-6 binds with a higher efficiency to the gp130 chain in vitro than does the mixture of IL-6 with sIL-6R (Kollet et al, 1999).
The IL-6R/IL-6 chimera has further been shown to induce the expression of myelin basic protein (MBP) and Po gene products MBP and Po RNAs and proteins in cultures of dorsal root ganglia (DRG) from 14 day old mouse embryos (Haggiag et al., 1999). MBP and Po proteins are normally induced during the final postnatal maturation of Schwann cells, and they are re-induced during nerve regeneration. The IL-6R/IL-6 chimera may thus have a role in neural myelination and regeneration.